Isolation of covalently closed circular DNA of high molecular weight from bacteria.
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Centrifugation, Density Gradient↗
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Manganese mutagenesis in yeast. A practical application of manganese for the induction of mitochondrial antibiotic-resistant mutations.
When yeast cells were incubated for 4 to 8 h in yeast extract-peptone-glucose medium, pH 6, containing 8 mM-manganese, and then plated on selective media, there was a strong induction of antibiotic-resistant mutations. Indirect evidence suggests that practically all resistant mutants selected were of independent origin. The analysis of manganese-induced resistant mutants showed that most were extranuclear, while those tested showed recombination with known mitochondrial markers. Our results suggest that manganese can be considered as a mutagen which specifically induces mitochondrial mutations in Saccharomyces cerevisiae.
Anti-Bacterial Agents↗
Effect of inorganic phosphate on acridine inhibition and plasmid curing in Escherichia coli.
Some mutants and stock strains of Escherichia coli K12 were sensitive to acriflavine in the presence of inorganic phosphate but were resistant to acriflavine in its absence. They mutated spontaneously to resistance to acriflavine plus phosphate. The synergistic effect of phosphate on acriflavine sensitivity was increased at high pH values. Genetic analysis suggested that the mutations occurred in the gene acrA. Electron microscopic observation suggested that the presence of acriflavine plus phosphate affected the structure of the plasma membrane and the cytoplasm under it. This structural alteration was not caused by acriflavine alone. Acridine orange plus phosphate can more effectively eliminate the plasmid F8-gal+ than acridine orange alone.
Acridines↗
Plasmid-mediated penicillin beta-lactamases in Pseudomonas aeruginosa.
A penicillin beta-lactamase (PCase) was extracted from Pseudomonas aeruginosa Rms139(+) and purified by means of column chromatography. The isoelectric point of Rms139 PCase was 5.7 and its molecular weight was 22,500 +/- 1,000. The optimal pH for the hydrolysis of benzylpenicillin was 7.0 to 7.5 and the optimal temperature was 45 C, with the PCase also showing high activity against carbenicillin. It is concluded that this enzyme is a new type of penicillin beta-lactamase different from the type I, II, or III R plasmid-mediated PCases reported previously.
Bacterial Proteins↗
Control of differentiation in streptomycetes: involvement of extrachromosomal deoxyribonucleic acid and glucose repression in aerial mycelia development.
When Streptomyces alboniger spores were grown in Hickey-Tresner broth containing 5 muM ethidium bromide, a high frequency of permanently cured aerial mycelia-negative (am-) colonies was recovered. The appearance an am- colonies was time dependent: a very low frequency (0.3%) at zero time, a maximum (9 to 21%) after 2 to 5 days of growth, and a decline again to low frequencies later in the growth cycle. On agar, cured am- colonies of S. alboniger still produced puromycin. The development of aerial mycelia in S. alboniger, S. scabies, and S. coelicolor was also sensitive to glucose repression. Colonies grown on Hickey-Tresner agar containing 2% glucose remained phenotypically am- throughout the observation period. Adenine (2.5 mM or greater), and to a lesser extent adenosine and guanosine, specifically reversed the repression. The accumulation of undissociated organic acids appears to be involved in glucose repression of aerial mycelia formation. However, this does not appear to be the case with puromycin production in S. alboniger; glucose repression was observed over the pH range 5.0 to 7.5.
Adenine↗
Regulation of enzyme formation in Klebsiella aerogenes by episomal glutamine synthetase of Escherichia coli.
We studied the physiology of cells of Klebsiella aerogenes containing the structural gene for glutamine synthetase (glnA) of Escherichia coli on an episome. The E. coli glutamine synthetase functioned in cells of K. aerogenes in a manner similar to that of the K. aerogenes enzyme: it allowed the level of histidase to increase and that of glutamate dehydrogenase to decrease during nitrogen-limited growth. The phenotype of mutations in the glnA site was restored to normal by the introduction of the episomal glnA+ gene. These results are consistent with the hypothesis that glutamine synthetase regulates the function of its own structural gene.
Ammonia-Lyases↗
Characterization of an endonuclease associated with the drug resistance plasmid pKM101.
An endonuclease was detected in strains of Salmonella typhimurium containing the drug resistance plasmid pKM101. The enzyme was not detectable in strains lacking this plasmid, but it was present in strains containing mutants of pKM101 that were no longer able to enhance host cell mutagenesis. The endonuclease had a molecular weight of roughly 75,000 and, at pH 7.0, was equally active on single-stranded and duplex deoxyribonucleic acid (DNA). The reaction with single-stranded DNA was optimal at pH 5.5, whereas with duplex DNA the optimum was pH 6.8. The enzyme required a divalent cation for activity, and it had no detectable exonuclease activity with single-stranded or duplex DNA. The endonuclease extensively degraded DNA with no apparent base specificity, forming 5'-phosphomonoester termini. Although characterization of the endonuclease has not revealed its function, the enzyme does not appear to be a restriction endonuclease.
DNA, Bacterial↗
[Resistance to antibiotics of bacteria involved in respiratory infections (author's transl)].
The present status of resistance to antibiotics of bacteria involved in respiratory infections is reviewed. Schematically it can outlined as follows. Streptococcus B-haemolyticus as well as Pneumococcus did not change their sensitivity to penicillin, but some strains are now resistant to tetracycline. Streptococcus viridans, Enterococcus and H. influenzae did not change substantially their sensitivity to antibiotics. Staphylococcus aureus is the bacterial species that always poses some problems with regard to antibiotic resistance. Due to the selection of strains penicillinase-producing because of the large use of penicillin, the most part of clinical isolates of staphylococci is now resistant to penicillin. In addition an increased number of strains resistant to the other antibiotics has been registered as soon as they has been introduced in therapy. The resistant strains spread in a particularly rapid way in hospital. The introduction in therapy of penicillinase-resistant penicillins constituted a remarkable advance in therapy of staphylococcal infection. However, there is now a growing number of indications about the emergence of methicillin resistant strains of staphylococci. On the other hand it must be recalled that since 1960 a marked reduction of incidence and mortality in severe staphylococcal infections has been noted. Incidence and mortality of respiratory infections due to Gram-negative bacilli is augmented particularly in connection with a larger use of immunosuppressive and antineoplastic therapies, of particular surgical or reanimation procedures, of intensive courses of antibiotic therapy etc. Emergence of Pseudomonas, Proteus, Serratia, Providencia, etc. infections poses many difficult problems of chemotherapy since these species are scarcely sensitive to antibiotics. Carbenicillin, cephalosporins, sisomicin, tobramycin, amikacin are the more recent drugs that alone or in combination may offer some chances of success in this field.
Anti-Bacterial Agents↗
Two distinct types of trimethoprim-resistant dihydrofolate reductase specified by R-plasmids of different compatibility groups.
R-Plasmids from a number of trimethoprim-resistant Escherichia coli and Citrobacter sp. were studied after transfer to E. coli K12 hosts. Each was found to specify a dihydrofolate reductase which was resistant to trimethoprim and Methotrexate, and which could be completely separated from the host chromosomal enzyme by gel filtration. Two distinct types of R-plasmid dihydrofolate reductases were identified. Type I enzymes, typified by the R483 enzyme previously described (Sköld, O., and Widh, A. (1974) J. Biol. Chem. 249, 4324-4325), are synthesized in amounts severalfold higher than the chromosomal enzyme. The 50% inhibitory concentrations (I50) of trimethoprim, Methotrexate, and aminopterin are increased several thousandfold over the corresponding values for the chromosomal enzyme. Type II R-plasmid dihydrofolate reductases are synthesized in about the same amount, or less, as the chromosomal enzyme, but are practically several hundredfold higher than those for the type I enzymes. Both types of R-plasmid dihydrofolate reductase showed little difference from the chromosomal enzyme in the binding of dihydrofolate, NADPH, folic acid, and 2,4-diaminopyrimidine.
Chromosomes, Bacterial↗
The evolutionary dynamics of extrachromosomal DNA in human cancers.
Oncogene amplification on extrachromosomal DNA (ecDNA) is a common event, driving aggressive tumor growth, drug resistance and shorter survival. Currently, the impact of nonchromosomal oncogene inheritance-random identity by descent-is poorly understood. Also unclear is the impact of ecDNA on somatic variation and selection. Here integrating theoretical models of random segregation, unbiased image analysis, CRISPR-based ecDNA tagging with live-cell imaging and CRISPR-C, we demonstrate that random ecDNA inheritance results in extensive intratumoral ecDNA copy number heterogeneity and rapid adaptation to metabolic stress and targeted treatment. Observed ecDNAs benefit host cell survival or growth and can change within a single cell cycle. ecDNA inheritance can predict, a priori, some of the aggressive features of ecDNA-containing cancers. These properties are facilitated by the ability of ecDNA to rapidly adapt genomes in a way that is not possible through chromosomal oncogene amplification. These results show how the nonchromosomal random inheritance pattern of ecDNA contributes to poor outcomes for patients with cancer.
Biological Evolution↗
Extrachromosomal DNA-Driven Oncogene Dosage Heterogeneity Promotes Rapid Adaptation to Therapy in MYCN-Amplified Cancers.
UNLABELLED: Extrachromosomal DNA (ecDNA) amplification enhances intercellular oncogene dosage variability and accelerates tumor evolution by violating foundational principles of genetic inheritance through its asymmetric mitotic segregation. Spotlighting high-risk neuroblastoma, we demonstrate how ecDNA amplification undermines the clinical efficacy of current therapies in cancers with extrachromosomal MYCN amplification. Integrating theoretical models of oncogene copy number-dependent fitness with single-cell ecDNA quantification and phenotype analyses, we reveal that ecDNA copy-number heterogeneity drives phenotypic diversity and determines treatment sensitivity through mechanisms unattainable by chromosomal oncogene amplification. We demonstrate that ecDNA copy number directly influences cell fate decisions in cancer cell lines, patient-derived xenografts, and primary neuroblastomas, illustrating how extrachromosomal oncogene dosage-driven phenotypic diversity offers a strong evolutionary advantage under therapeutic pressure. Furthermore, we identify senescent cells with reduced ecDNA copy numbers as a source of treatment resistance in neuroblastomas and outline a strategy for their targeted elimination to improve the treatment of MYCN-amplified cancers. SIGNIFICANCE: ecDNA-driven tumor genome evolution provides a major challenge to curative cancer therapies. We demonstrate that ecDNA copy-number dynamics drives treatment resistance by promoting oncogene dosage-dependent phenotypic heterogeneity in MYCN-amplified cancers. Exploiting phenotype-specific vulnerabilities of ecDNA cells, therefore, presents a powerful strategy to overcome treatment resistance. See related commentary by Korsah, p. 1979.
Humans↗